TITLE: A Process for Conditioning Grain

FIELD OF THE INVENTION

The present invention relates to a process for conditioning grain prior to the milling of grain. The invention furthermore relates to compositions for use in such a process.

BACKGROUND OF THE INVENTION

The milling of grain into flour generally comprises a number of steps of which the first step is the conditioning of the grain to prepare it for the actual grinding and separation processes that ultimately provide for a number of final products, such as flour(s), and bran(s).

A number of conditioning processes have been developed for the industrial milling of wheat. The object of conditioning, the central feature of which is the addition of water to grain, is to modify the wheat kernel so that milling can be performed under optimal conditions. Water is added, usually to obtain a moisture content of 16% and, after storage, which generally lasts several hours, additional water is often added before milling. The optimal amount of water as well as tempering time depends on the properties of the grain. Conditioning influences not only milling quality but also the quality of the end flour product.

The primary aim of conditioning is to change the mechanical characteristics of the different tissues of the kernel and thereby improve the separability of the endosperm from the outer layers of the grain, notably the bran. The addition of water also triggers a number of biochemical reactions in the kernel, thereby modifying characteristics of its components. These modifications can be amplified by increasing the temperature and the moisture content.

It has been described to use enzymes (cellulase, hemicellulase, lipase and protease/amylase) in a wheat separation process. It was concluded that cellulases and hemicellulases improve the processing properties of wheat and increase the yield of gluten and starch. The hemicellulase used was a xylanase preparation containing significant side-activities including amylase and protease activity (Weegels et al., Starch/Starke, 44 No. 2, pp. 44-48, 1992).

It is known that xylanases are capable of degrading wheat flour and other plant derived materials into a number of different degradation products. Xylanases purified from a strain of the fungal species Aspergillus awamori have been described in a number of references, e.g.

Kormelink, FJ. M. et al. Journal of Biotechnology., 27, pp. 249-265 (1993), disclosing physicochemical and kinetic characteristics of three A. awamori endo-xylanases.

The degradation products obtained by use of such A. awamori xylanases in the enzymatic degradation of various plant materials including rice bran, oat spelts, wheat flour, larch

wood and birch wood are described by Kormelink, FJ. M. and Voragen, A.G.J. , Appl. Microbiol. Biotechnol., 38, pp. 688-695 (1993); H. Gruppen et al., Carbohydr. Res. 233, pp. 45-64 (1992).

It has been disclosed to use a xylanase preparation, alone and in combination with other enzymes, for reducing the viscosity of a plant material, especially in wheat and corn starch processing, and for wheat separation into starch-containing and protein-containing components (WO 99/21656; Novozymes A/S).

None of the above references mentions that xylanases may be used in a pre-milling grain conditioning process to increase the flour yield, and how to select a xylanase for that particular purpose. The commercial grain milling industry is a highly competitive business, where even incremental flour yield improvements are highly interesting. There is a constant need for improved processes that achieve greater flour yields.

SUMMARY OF THE INVENTION It has now surprisingly been found that a treatment of grain, prior to milling of the grain, with an enzyme composition comprising substantial xylanase activity improves the subsequent grain milling process in that it provides an increase in the yield of flour, and/or a reduction of the conditioning time, and/or an improvement of the properties of the flour/bran produced.

Accordingly, in a first aspect the invention relates to a process for pre-milling conditioning of a grain, wherein prior to milling of the grain, the grain is treated with an enzyme composition comprising a xylanase, preferably from the xylanase family 10. The xylanase family 10 is well-known, see for instance the review by Collins T et al. Xylanases, xylanase families and extremophilic xylanases, FEMS Microbiol Rev. (2005) Jan;29(1 ):3-23.

DETAILED DESCRIPTION OF THE INVENTION

According to the invention a process for pre-milling conditioning of a grain is provided, wherein prior to milling of the grain, the grain is treated with an enzyme composition comprising a xylanase.

It has been found that the process of the invention enhances the yield of flour obtained thereby increasing the value of product, as well as improving the rheological properties of the flour, thereby increasing the usefulness of the product so obtained.

Preferably, the process of the invention provides an enhanced yield of flour.

Examples of such increased flour yields, obtained by a process of the invention, are demonstrated in working example 1 below. A further advantage is that it is possible to maintain a certain level of flour yield

while reducing the necessary grain conditioning time significantly, thereby freeing capacity in the milling plant.

Examples of enzyme preparations according to the invention are the enzyme compositions A, B, or C, which are described in the Materials and Methods. These enzyme

5 compositions are mixtures of a highly concentrated xylanase together with enzyme activities selected from the group comprising proteases, cellulases, pectinases, hemicellulases, glucanases, β-glucanases, glucose oxidases, laccases, and amylases.

Among the activities mentioned above it has been found that the most important ones are proteases, xylanases, endoglucanases, pectinase, hemicellulase, cellulase, glucose 10 oxidase, and/or laccase activities.

Generally, the preferred conditioning time depends on the actual type of grain and in particular whether it is a soft, mid hard or hard grain.

In the present context a "soft grain" denotes a grain with the following average characteristics: W=80-150, P/L=0.2-0.5 as measured on an Alveograph (W: strength; P: 15 Tenacity; L: extensibility); a "mid hard grain" denotes a grain with the following average characteristics W=150-300, P/L= 0.5-0.8 as measured on an Alveograph (W: strength; P: Tenacity; L: extensibility); and a "hard grain" denotes a grain with the following average characteristics: W=300- 20 400, P/L= 0.8-1 as measured on an Alveograph (W: strength; P: Tenacity; L: extensibility).

Accordingly, a preferred embodiment relates to a process of the first aspect, wherein said grain is a soft grain, a mid hard grain, or a hard grain; preferably said grain is a hard grain, as defined herein.

In a preferred embodiment, the grain is selected from the group comprising cereal 25 grain (e.g., wheat, oat, corn). In another preferred embodiment, the grain is selected from the group comprising wheat, barley, rye, oat, corn, rice, and legume grains, such as alfalfa, soy bean, and rape seed, soy bean, jatrofa.

It is also preferred that the grain has a humidity content of 5% to 50%; preferably from 10% to 40%; or most preferably from 12% to 30%.

30 In a preferred embodiment of the process of the first aspect of the invention, said grain is treated for a period of time from 1 - 48 hours; preferably from 1 - 36 hours; most preferably from 1 - 24 hours; and most preferably from 1 - 12 hours.

It is preferred in the process, the grain is treated with the enzyme composition in a dosage having xylanase activity corresponding to at least 10,000 FXU per ton grain, preferably

35 at least 15,000 FXU per ton grain, 20,000 FXU per ton grain, 25,000 FXU per ton grain, 50,000

FXU per ton grain, 75,000 FXU per ton grain, or most preferably at least 100,000 FXU per ton grain, as defined herein; preferably said grain is treated with the enzyme composition in a dosage having xylanase activity corresponding to at least 10,000 FXU per ton grain, preferably at least 15,000 FXU per ton grain, 20,000 FXU per ton grain, 25,000 FXU per ton grain, 50,000 FXU per ton grain, 75,000 FXU per ton grain, or most preferably at least 100,000 FXU per ton grain; in combination with alpha-amylase activity corresponding to at least 50,000 KNU per ton grain and cellulase activity corresponding to at least 80,000 EGU per ton grain, as defined herein; or preferably said grain is treated with the enzyme composition in a dosage having xylanase activity corresponding to at least 170,000 FXU per ton grain in combination with alpha- amylase activity corresponding to at least 80,000 KNU per ton grain, as defined herein; or preferably said grain is treated with the enzyme composition in a dosage having xylanase activity corresponding to at least 250,000 FXU per ton grain in combination with cellulase activity corresponding to at least 50,000 EGU per ton grain, as defined herein. The dosage FXU, KNU and EGU are standard activity units for xylanase, alpha-amylase and cellulase, respectively, and can be determined as described in the Materials and Methods.

In a preferred embodiment, the xylanase exhibits a WSPS per mg protein added of at least 0.06, and/or a WSPU per mg protein added of at least 15.

In the present context, WSPU (Water Soluble Pentosan Unit) is defined as the activity of the xylanase preparation per mg protein on water soluble pentosans and WIPU (Water Insoluble Units) as the activity on water insoluble pentosans per mg protein where the activities are measured as reducing sugars and where the protein is determined according to Kjeldahl, cf the Materials and Methods section herein. The production of soluble and insoluble pentosans, respectively, is described in the Materials and Methods section below. In Example 2 an assay for determining WSPU and WIPU is described. WSPS is the ratio between WSPU and WIPU.

In the present context, the term "protein added" is intended to be the amount of protein comprising xylanolytic activity, which is recovered from a fermentation broth in which the enzyme has been produced, and which subsequently is used for the present purpose. Thus, when assessing the WSPS and WSPU values as defined herein of a given enzyme, the term "mg protein" refer to the mg protein associated with the enzyme when recovered from the fermentation broth and without any inert or non-xylanolytic protein.

It is preferred that the xylanase preparation to be used for the present purpose is one having a WSPS which is higher than 0.06. Preferably the WSPS added is in the range of 0.06 to at most 10.0, more preferably of at least 0.7 to at most 8.0, still more preferably between 0.9 and 6.0, especially of at least 1.5 to 4.0, and/or a WSPU per mg protein added which is

higher than 15, such as 25 or more. Preferably the WSPU per mg protein added is at least 100 to at most 150.000, more preferably at least 130 to at most 120.000, such as at least 160 to at most 100.000, more preferably of at least 300 to at most 90.000, and still more preferably of at least 20.000 to at most 85.000. In Example 2 the WSPS and WSPU values of the H. insolens xylanase is compared with that of xylanase Il (of family 10).

Another preferred embodiment relates to the process of the first aspect, wherein said enzyme composition also comprises at least one enzyme activity selected from the group of enzyme activities consisting of protease, cellulase, pectinase, hemicellulase, glucanase, β- glucanase, glucose oxidase, laccase, and amylase.

It is preferred that said enzyme composition comprises an alpha-amylase; a cellulase; or both an alpha-amylase and a cellulase; preferably the alpha-amylase is an 1 ,4- alpha-D-glucan glucano-hydrolase (EC 3.2.1.1 ) from Bacillus amyloliquefaciens; preferably BAN® 480 L (Novozymes A/S, Denmark); and preferably the cellulase is from Tricoderma longibrachiatum (formerly known as reesei); preferably Celluclast® 1.5 L (Novozymes A/S, Denmark).

Three particular enzyme compositions according to the invention are disclosed and applied in the example below, and those are of course also preferred in an embodiment of the process of the first aspect, wherein said enzyme composition comprises the enzyme composition A, B, or C, as defined herein under the Materials and Methods section.

Regarding the xylanase in the process of the first aspect, it is preferred that said xylanase comprises a xylanase from family 10, preferably xylanase Il from the fungal species Aspergillus aculeatus CBS 101.43 the amino acid sequence and DNA coding sequence of which are shown in SEQ ID NOs 5 and 2, respectively, in WO 94/21785, which sequences are incorporated herein by reference in their totality.

The DNA sequence encoding Xylanase Il is shown in SEQ ID No. 2 of WO 94/21785 and the corresponding amino acid sequence in SEQ ID No. 5. It is contemplated that xylanases exhibiting high homology or identity to xylanase Il may have a similar activity pattern as xylanase Il and thus be useful for the present purpose. Accordingly, in a particularly preferred embodiment the xylanase to be used in the present invention is one, which i) is encoded by the DNA sequence shown in SEQ ID No. 2 of WO 94/21785 or an analogue thereof encoding a homologue of Xylanase II, ii) comprises the amino acid sequence shown in SEQ ID No. 5 of WO 94/21785 or a homologous sequence thereof, and/or iii) is immunologically reactive with an antibody raised against a purified xylanase

derived from Aspergillus aculeatus, CBS 101.43.

In the present context, the term "homologue" is intended to indicate a polypeptide exhibiting xylanase activity, encoded by a DNA sequence hybridizing with an oligonucleotide probe prepared on the basis of the DNA sequence coding Xylanase Il enzyme under certain specified conditions (such as presoaking in 5xSSC and prehybridizing for 1 h at ~40°C in a solution of 5xSSC, 5xDenhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50 microg of denatured sonicated calf thymus DNA, followed by hybridization in the same solution supplemented with 50 microCi 32-P-dCTP labelled probe for 18 h at ~40°C followed by washing three times in 2xSSC, 0.2% SDS at 40 ^° C for 30 minutes). The term "homologue" is intended to include modifications of the DNA sequence

SEQ ID No. 2 of WO 94/21785, such as nucleotide substitutions which do not give rise to another amino acid sequence of the xylanase, but which correspond to the codon usage of the host organism into which the DNA construct is introduced or nucleotide substitutions which do give rise to a different amino acid sequence and therefore, possibly, a different protein structure which might give rise to a xylanase mutant with different properties than the native enzyme. Other examples of possible modifications are insertion of one or more codons into the sequence, addition of one or more codons at either end of the sequence, or deletion of one or more codons at either end or within the sequence. It will be understood that the homologue of xylanase Il defined herein may comprise a number of different amino acid residues as long as the xylanase activity is as defined herein.

The present invention is also directed to the use of xylanases which have at least 70% identity to the DNA sequence shown in SEQ ID No. 2 of WO 94/21785 or a substantial part thereof, such as, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the DNA sequence of SEQ ID No. 2 of WO 94/21785 or a substantial part thereof encoding a polypeptide with xylanase activity.

The present invention is also directed to xyalanses which have at least 70% identity to the amino acid sequence shown in SEQ ID No. 5 of WO 94/21785, such as, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID No. 5 of WO 94/21785. The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "identity." For purposes of the present invention, the degree of homology between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. MoI. Biol. 48: 443- 453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16:

276-277; emboss.org), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues x 100)/(l_ength of Alignment - Total Number of Gaps in Alignment)

For purposes of the present invention, the degree of identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra; emboss.org), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

The production of Xylanase Il and further characterization thereof is apparent from the disclosure presented in WO 94/21785. The xylanase preparation to be used herein may be obtained from a microorganism in question by use of any suitable technique. For instance, a xylanase preparation may be obtained by fermentation of a microorganism and subsequent isolation of the enzyme by a method known in the art, but more preferably by use of recombinant DNA techniques known in the art. Such method normally comprises cultivation of a host cell transformed with a recombinant DNA vector capable of expressing and carrying a DNA sequence encoding the xylanase in question, in a culture medium under conditions permitting the expression of the enzyme and recovering the enzyme from the culture.

Within the scope of the invention are any enzyme preparations prepared from microbially derived mono-component enzymes (i.e. substantially without any side activity). The DNA sequence encoding the xylanase to be used may be of any origin, e.g. a cDNA sequence, a genomic sequence, a synthetic sequence or any combination thereof. The preparation of a xylanase suited for the present purpose is described in detail in WO 94/21785.

It has been found, that the flour yield increase effect obtained in the grain treatment process of the invention may be considerably improved when the xylanase is used together with a cellulase and/or an amylase.

Accordingly, the xylanase preparation to be used in the present invention may comprise a cellulase. The cellulase is preferably of microbial origin, such as derivable from a strain of a filamentous fungus (e.g. Aspergillus, Trichoderma, Humicola, Fusarium). Specific examples of cellulases suitable for the present purpose include the endo-glucanase (endo- glucanase I) obtainable from H. insolens and further defined by the amino acid sequence of fig. 14 in WO 91/17244 and the 43 kD H. insolens endoglucanase described in WO 91/17243.

Commercially available cellulase preparations which may be used in combination with a xylanase as described herein include Celluclast® 1.5 L (available from Novozymes A/S), Spezyme® CP (available from Genencor, USA) and Rohament® 7069 W (available from Rohm, Germany).

Yet another preferred embodiment relates to the process of the first aspect, wherein said treatment is performed at a temperature between 5 ^° C and 60 ⁰ C, preferably between 10 ⁰ C and 40°C, and more preferably between 20 ⁰ C and 30°C.

It is also preferred that the treatment according to the process of the first aspect is performed by the addition of said enzyme preparation in an amount of between 1 - 50,000 g enzyme preparation per ton of grain; preferably in an amount of between 10 - 25,000 g enzyme composition per ton of grain; and most preferably in an amount of between 50 - 2,000 g enzyme composition per ton of grain.

In a preferred embodiment of the first aspect, the ratio between FXU and EGU is at least 0.5, more preferably at least 1.0, and most preferably at least 2.0.

Finally, it is preferred that a flour yield increase is obtained after milling grain treated in the process of the first aspect of the invention, as compared to the yield of flour obtained from milling grain which has gone through the same pre-milling grain conditioning process without the enzyme preparation; preferably the flour yield increase is at least 1%, more preferably 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and most preferably at least 10%.

Examples of industries which advantageously may use a process of the invention are industries such as the i) the milling industry for e.g. getting a higher yield of flour; ii) the starch industry to e.g. get a higher yield of flour or higher purity of protein and/or starch fractions, or to modify the flour composition such as fibre enrichment of the flour, and iiii) the bioethanol industry to e.g. get higher ethanol yields after fermentation or lower viscosity during whole process for ethanol production from grain.

Accordingly, further embodiments of the invention relate to use of a process for the conditioning of grain, according to the invention, in the milling industry; the brewing industry; the

starch industry; and/or the bioethanol industry.

EXAMPLES Materials and Methods Grain: Wheat that has a humidity or moisture content below 16% as commercially available.

Enzyme preparations:

• Viscozyme® L is a commercially available liquid beta-glucanase (endo-1 ,3(4)-) enzyme preparation (Novozymes A/S) with side activities of xylanase, cellulase, and hemicellulase. The declared activity is 100 FBG/g (fungal beta-glucanase units) and the preparation has been measured to 24 FXU/g (fungal xylanase units) and 113 EGU/g (endu glucanase units). Density approx. 1.10 g/ml.

• Celluclast® 1.5 L is a commercially available liquid cellulase enzyme preparation (Novozymes A/S). The declared activity is 700 EGU/g (endu glucanase units) and the preparation has been measured to 50 FXU/g. Density approx. 1.20 g/ml.

• Shearzyme® 2X is a commercially available liquid xylanase enzyme preparation (Novozymes A/S). The declared activity is 1 ,000 FXU-S/g (fungal xylanase units). Density approx. 1.10 g/ml.

• BAN® 480 L - Bacterial Amylase Novo is a commercially available liquid alpha-amylase (1 ,4-alpha-D-glucan glucano-hydrolase (EC 3.2.1.1)) enzyme preparation (Novozymes

A/S). The declared activity is 480 KNU/g (kiloNovo units). Density approx. 1.2 g/ml.

• A is an enzyme blend of BAN® 480 L, Shearzyme® 2X, and Celluclast® 1.5 L in the volumetric proportion 1 :1 :1 , which results in a theoretical activity of approx. 160 KNU/g; 333 FXU/g; 233 EGU/g. Density approx. 1.17 g/ml. • B is an enzyme blend of BAN® 480 L and Shearzyme® 2X in the volumetric proportion 1 :1 , which results in a theoretical activity of approx. 240 KNU/g; 500 FXU/g. Density approx. 1.15 g/ml.

• C is an enzyme blend of Celluclast® 1.5 L and Shearzyme® 2X in the volumetric proportion 1 :3, which results in a theoretical activity of approx. 175 EGU/g; 750 FXU/g. Density approx. 1.13 g/ml.

Productions of soluble pentosan (WSP) from wheat flour

100 kg common wheat flour was suspended in 300 kg cold water. After stirring for 1 hour the sludge was removed using a decanter. The resulting supernatant was then subjected to enzymatic treatments to remove starch and protein. After adjusting pH to 6.5 and the

temperature to 90 ⁰ C was first added 2% (of d.m.) of Termamyl® 120L with 90 min. of stirring, followed by a similar treatment with 3% (of d.m.) AMG® 300L at pH 4.6 and 60 ⁰ C for 120 min to hydrolyse the starch fraction. Finally, the supernatant was treated with 1% (of d.m.) of Alcalase® 2.4L at pH 8.0 and 55°C for 120 min under constant stirring. After hydrolysis of starch and protein the product was filtered on filterpress using Seitzfilter K250 to remove residual insoluble material, and the supernatant ultrafiltrated using a 10,000 MW cut-off membrane to remove products from the starch and protein hydrolysis. The retentate was further diafiltrated until 0 ⁰ BRlX was reached. The product was concentrated by evaporation on a Luwa Evaporater and finally freeze dried.

Production of insoluble pentosan (WIP) from wheat flour

150 kg of common wheat flour was suspended in 450 kg of cold water. The suspension was heated to 60°C and 600 g of Termamyl® 120L were added. After heating to 95°C resulting in gelatinization of the starch fraction, the suspension was cooled to 60 ⁰ C with continued hydrolysis for 180 min. After adjusting the pH to 8.0 using NaOH 300 g of Alcalase® 2.4L were added. During hydrolysis of protein under constant stirring, the pH was maintained between 7.5 and 8.0 titrating with NaOH. The hydrolysis was continued for 120 min. the precipitate was recovered after centrifugation, washed with water once and then further washed on a 35 μm sieve with cold water to remove all residual soluble material. To the resulting insoluble material up to 20 I of water was added, heated to 60 ⁰ C and after an adjustment of the pH to 8.0 with NaOH 100 g of Alcalase® 2.4L were added. The hydrolysis and NaOH-titration were continued until no further drop in pH was observed. The material was then washed again on a 35 μm sieve until all soluble material was removed and, finally, freeze dried.

The activity of the xylanase to be used in the present invention is measured by the release of reducing sugars from soluble pentosan (diluted 25 x after incubation), and insoluble pentosan (diluted 5 x after incubation).

0.5% of water soluble or water insoluble pentosans produced as described above is dissolved or suspended in a 0.1 M citrate/phosphate buffer, pH 6.0. Per sample 0.9 ml of the substrate is mixed with 0.1 ml of enzyme solution. The substrate is held on ice before and during the mixing of enzyme and substrate. Incubation takes place at 40 ⁰ C for 15 min. whereafter the enzyme is denaturated at 100°C for 5. min. When the samples are cooled the soluble pentosan solutions are diluted 25 times while the insoluble solutions are diluted 5 times. Then, reducing sugars are determined by reaction, in microtiter plates, with a PHBAH reagent comprising 0.15 g of para hydroxy benzoic acid hydrazide (Sigma H-9882), 0.50 g of potassium-sodium tartrate (Merck 8087) and 2% NaOH solution up to 10.0 ml. Results of blanks are subtracted. Xylose is

used as a standard. The reducing sugars may be used in determining WSPU and WSPS.

Protein assay - Kjeldahl

The assay was performed by use of a Tecator Digestor and distillation unit type 1003. The samples to be analyzed are transferred to Kjeldahl tubes, for fluid samples approx. 1.5 g and for freeze-dried samples approx. 0.1 g. To the samples are added: a) 3.0 ml sulphuric acid (cone. H2SO4) b) 1.5 ml hydrogen peroxide (32% H2O2) c) 1 Kjeltab (Se + K2SO4) If the samples foam after addition of the chemicals they are not destructed till the next day. Under normal circumstances the samples are placed in the destruction block after 10 min. The block temperature is set to 370 ⁰ C. When the samples are clear or faintly yellow, they are removed. The destruction usually takes /4-1 hour according to the composition of the sample. The samples are cooled at room temperature for about 20 min. Then they are ready for distillation.

The samples are distilled in 25 ml 2% boric acid containing Kjeldahl indicator (A: 0.12 g Methylene blue in 100 ml 96% alcohol and B: 0.125 g Methylene red in 100 ml 96% alcohol, A and B being mixed in the proportion 1A:2B). The destructed sample is mixed with 32.5% NaOH. The ammonium is distilled into the 2% boric acid which is then titrated with 0.1 N HCI until pH reaches 4.85.

Determination of endo-glucanase activity (EGU)

The fermentation broths are analyzed by vibration viscosimetry on CMC at pH 6.0.

More specifically, a substrate solution containing 34.0 g/l CMC (Blanose Aqualon) in 0.1 M phosphate buffer, pH 6.0 is prepared. The enzyme sample to be analyzed is dissolved in the same buffer. 14 ml substrate solution and 0.5 ml enzyme solution are mixed and transferred to a vibration viscosimeter (e.g. MIVI 3000 available from Sofraser, France) thermostated at 40 ⁰ C.

One EGU is defined as the amount of enzyme that reduces the viscosity to one half under these conditions. The amount of enzyme sample should be adjusted to provide 0.01-0.02 EGU/ml in the reaction mixture. The arch standard is defined as 880 EGU/g.

Determination of xylanase activity (FXU)

The endo-xylanase activity is determined by an assay, in which the xylanase sample is incubated with a remazol-xylan substrate (4-O-methyl-D-glucurono-D-xylan dyed with Remazol Brilliant Blue R, Fluka), pH 6.0. The incubation is performed at 50 ⁰ C for 30 min. The background

of non-degraded dyed substrate is precipitated by ethanol. The remaining blue colour in the supernatant is determined spectrophotometrically at 585 nm and is proportional to the endoxylanase activity. The endoxylanase activity of the sample is determined relatively to an enzyme standard. The assay is further described in the publication AF 293.6/1 -GB, available upon request from Novozymes A/S, Denmark, which folder is hereby included by reference.

Determination of amylase activity

Acid fungal alpha-amylase activity may be measured in KNU (Kilo Novo alpha Amylase Unit). One Kilo Novo alpha Amylase Unit (KNU) is defined as the amount of enzyme which, under standard conditions (i.e. at 37°C+/- 0.05; 0.0003 M Ca2+; and pH 5.6) dextrinizes 5.26 g starch dry substance Merck Amylum solubile.

A folder AF 9/6 describing this analytical method in more detail is available upon request to Novozymes A/S, Denmark, which folder is hereby included by reference.

Assessment of flour quality:

Flour Yields: A CHOPIN™ Laboratory Mill (model CD1 ) available from Chopin, France was used for the milling of the grain after conditioning. The mill separates the ground wheat into two fractions, flour and bran.

Example 1

Hard grains were treated separately with different enzyme preparations for 2, 12, and 24 hours prior to determining the flour yields in the laboratory mill. The grains were treated with two commercially available standard enzyme preparations, Viscozyme® L and Celluclast® 1.5 L (both Novozymes A/S), as well as with three blended enzyme compositions comprising an xylanase according to the invention: Composition A, B, or C.

Lab conditioning trials were carried out on Hard wheat (Yecora) from Spain, using 500 g of each, having an initial moisture of 10.70%. Water was added 29 ml to the hard wheat. The trials were kept in plastic bottles resting for 2, 12, and 24 hours.

All enzyme preparations were added in a dosage of 300 ml per ton grain. 300 ml of each of the three NZ enzyme compositions of the invention corresponds to 351 g of composition A; 345 g of composition B; or 339 g of composition C. Expressed in xylanase units per ton grain in the pre-milling conditioning treatment, the dosage of 300 ml of each enzyme composition corresponds to 116,883 FXU/t of composition A, 172,500 FXU/t of B; or 254,250 FXU/t of composition C. Analogously, the levels of xylanase activity in the prior art enzyme treatments with Viscozyme ^® L or Celluclast ^® 1.5L were 7,920 FXU/t and 18,000 FXU/t, respectively.

The resulting relative flour yield improvements from hard grain, as compared to where no enzyme was added in the pre-milling grain conditioning, are summarized in table 1 below. Clearly, pre-milling treatment of the hard grains with the three blended NZ enzyme compositions according to the invention provides significantly improved flour yields, especially at the two shorter conditioning times of 2 and 12 hours, when compared to a treatment with either of the two commercially available enzyme preparations Viscozyme ^® L and Celluclast ^® 1.5 L.

Table 1.

Example 2. WSPS and WSPU determinations In determining the WSPS and WSPU the protein content of the enzyme added determined according to Kjeldahl and the activities of the enzymes upon water insoluble (WIP) and soluble pentosans (WSP) produced as described previously measured as reducing sugars must be determined. The WSPS and WSPU values determined for the preparations shown in table 2.

Table 2.

The WSPU is the activity on WSP per mg protein pr. kg flour and the WSPS is the ratio WSPU/WIPU pr. kg flour. As can be seen Xylanase Il exhibits a remarkable high WSPU and WSPS which reflexes the degradation of water soluble pentosans at a high rate and a low degradation at insoluble pentosans compared to protein addition.